Di-(2-ethylhexyl) phthalate (DEHP), a commonly used plasticizer, has an adjuvant effect in combination with ovalbumin (OVA). The adjuvant effect of DEHP has already been verified in our previous studies. In this study, to further investigate whether thymic stromal lymphopoietin (TSLP) was involved in the DEHP-adjuvant effect, DEHP was administered through a daily gavage exposure route. Mice were sensitized with ovalbumin (OVA) to trigger allergic responses, and an anti-TSLP monoclonal antibody was used to neutralize the effect of TSLP. Biomarkers including cytokines in bronchoalveolar lavage fluid (BALF), serum total IgE and TSLP content in the lung were detected. In addition, airway hyperreactivity and lung sections were examined. Collectively, these data indicated a salient Th2 response which was characterized by the upregulation of Th2-type cytokines, such as interleukin 4 (IL-4), IL-5 and IL-13. Moreover, the eosinophil number in BALF and the eosinophil cationic protein (ECP) in the lung were seen to have increased significantly. However, neutralization of TSLP with an anti-TSLP mAb reversed the adjuvant effect of DEHP on airway inflammation, structural alterations in the airway wall and increased airway hyperresponsiveness (AHR) to methacholine induced by the OVA allergen, suggesting that TSLP was an effective target site for suppressing the adjuvant effect of DEHP co-exposure. In recent years, reports of about the illegal use of the phthalate plasticizer, di-(2-ethylhexyl) phthalate (DEHP), have raised concerns among medical institutions, regulatory agencies and the public. DEHP is widely used as a plasticizer in polyvinyl chloride from which it can leach and thence be absorbed by the human body. DEHP exposure is associated with the development of wheezing and allergic airway diseases, and has been shown to contribute to asthma occurrence in Sweden –2. Addit+ OVA T cells [Thymic stromal lymphopoietin (TSLP) is first isolated from the supernatant of a murine thymic stromal cell line, initially identified as a pre-B-cell . As an a T cells . Neutral T cells .Diisononyl phthalate (DINP) has similar physical and chemical properties as DEHP, and can aggravate AD-like skin lesions via a TSLP-mediated pathway . DibutylAllergic asthma is a common chronic disease that occurs in the bronchial epithelium, and its representative symptoms include lung inflammation, airway hyperresponsiveness (AHR) and mucus overproduction . EvidencIn this study, a chronic OVA-induced mouse model of asthma was used to investigate the role of TSLP in the DEHP-adjuvant effect on the coallergen (OVA). Local treatment by anti-TSLP mAb was used to block the effect of TSLP, which was the link between DEHP co-exposure and the on-going adjuvant effect on allergic inflammation.All experimental procedures were approved by the Office of Scientific Research Management of Central China Normal University, and the Application for the Use of Animals was certified on Nov. 8, 2011  . AccordiSPF male Balb/c mice (5–6 weeks) were purchased from the Hubei experimental animal centre  and housed in an environment with 12-hour light-dark cycle, 20–25°C and 50–70% humidity. OVA-free food and tap water were offered ad libitum. Prior to administration, the mice were quarantined for at least a week.2A by intraperitoneal (i.p.) injection (20 μg per mouse) (IgG control group). Briefly, mice were sensitized with saline or 50 μg OVA plus 1.75 mg Al(OH)3 in 300 μl saline on day 20 (by i.p. injection), day 34, day 41 and day 48 (by subcutaneous injection). For the TSLP blocking experiment, the mice received 20 μg of anti-TSLP monoclonal antibody (mAb)  or the isotype control IgG2A via i.p. injection  60 minutes prior to OVA sensitization, the dose of anti-TSLP mAb being the same as that in a previous study [The schematic diagram of the treatment schedule is shown in us study . All micBlood samples were collected by heart puncture. After centrifugation  the serum was collected and stored at -70°C before analysis. The lowest detection limit of total IgE was 0.156 ng/ml, and the sensitivities of OVA-specific IgE and OVA-specific IgG1 assays were both 0.1 ng/ml. All operations were conducted according to the manufacturer’s instructions.Cytokine levels were detected using ELISA kits  according to the kits’ instructions. The lowest detection limit of IL-4 and IFN-γ were both 15 pg/ml, and the sensitivities of IL-5 and IL-13 were 5 pg/ml and 4 pg/ml respectively.Lungs were lavaged three times with 0.5 ml, 0.6 ml and 0.7 ml ice-cold saline using a tracheal cannula and a 1 ml polyethylene syringe. The volumes of qualified samples were 1.3 to 1.5 ml. The supernatant of BALF samples was collected after centrifugation  and stored  for analysis. The pellets of BALF samples were then resuspended with cold sterile saline, and counted using a haematology analyser . The number of eosinophils (EOS) and the ratio of neutrophils (NEUT) in BALF were recorded.i.p. injection. When the mice showed no response to a pin prick acupuncture on their feet, the skin of the neck was carefully incised, the trachea was intubated, and quickly connected to a small computer-controlled ventilator via the tracheal cannula; According to the manufacturers’ instructions of the AniRes2005 lung function system , the ratio of expiration/inspiration was preset at 1.5:1 and the actual respiratory rate was preset at 90 breaths/min. While the volume of air used in the mechanical ventilator was 8 ml/kg, and for 20 g mouse the volume air was 0.16 ml per breath. The AniRes2005 software was then run. When all the parameters were stable, methacholine  was injected into the jugular vein at doses of 0.025, 0.05, 0.1 and 0.2 mg/kg successively every 5 minutes using the injector needle through the fixed tracheal cannula. The software recorded the inspiratory and expiratory resistance R-areas (Ri and Re) and the lung dynamic compliance (Cldyn). The R-area (100 Pa·ml-1) represents the area between the peak curve of Ri or Re. The baseline level was recorded during the 250s time period after MCH entered the vein, while the lowest value of Cldyn (ml·1000 Pa-1) was analysed to quantitatively assess lung compliance [The airway hyperresponsiveness (AHR) measurement was conducted 24 h after the final aerosol challenge using an AniRes2005 lung function system  according to the manufacturer’s instructions. Mice were anesthetised with pentobarbital sodium (Sigma) at a dose of 100 mg/kg by mpliance .Lungs were excised from the thoracic cavity, inflated, fixed with 4% neutral buffered polyformaldehyde and then cut into slices. Tissues were embedded in paraffin and stained with hematoxylin&eosin (H&E), Periodic acid-Schiff (PAS), or Masson’s trichrome (MT). Pictures were taken using a DM 4000B Microscope (Leica), and analyzed with Image-Pro Plus 6.0 to determine the average optical density (AOI) of the PAS and MT stained sections.TSLP levels in lung and BALF were measured with the mouse TSLP ELISA kit . According to the manufacturers’ instructions of eBioscience mouse TSLP ELISA kit, the functional minimum sensitivity is 16 pg/ml and the standard curve range is 16–2000 pg/ml.p-value of less than 0.05 was considered to be a significant difference.Analysis of variance (ANOVA) was used for statistical analysis. The data were presented as the mean ± standard error of the mean (SEM). One-way analysis of variance (one-way ANOVA) was used to determine the levels of difference between groups. A p<0.01) (p<0.01). Compared with the DEHP+OVA group, we observed that neutralization of anti-TSLP mAb significantly inhibited the elevation of total IgE (p<0.01), OVA-sIgE (p<0.01) and OVA-sIgG1 (p<0.01)  . It was (p<0.01) .p<0.05 or p<0.01), the DEHP+OVA group (p<0.01), the anti-TSLP group (p<0.05 or p<0.01) and the IgG control group (p<0.01) (p<0.01) in the DEHP+OVA group when compared to the OVA group. Furthermore, pre-treatment with anti-TSLP mAb diminished the increase of IL-4, IL-5 and IL-13 concentrations in BALF (p<0.01), as well as there being an apparent increase in the ratios of IFN-γ and IL-4 in BALF (p<0.01) in the anti-TSLP group when compared to the DEHP+OVA group  and the ratio of Th1 cytokine (IFN-γ) and IL-4 in BALF were detected using ELISA kits to assess the nature of the lymphocyte cytokine response to DEHP and/or OVA . IL-4, I(p<0.01) . In addiVA group .+ cells into the airway, we counted the number of inflammatory cells in BALF. The data on cell count demonstrated that blocking TSLP with anti-TSLP mAb effectively eliminated the DEHP-adjuvant effect on elevated eosinophil counts and on the percentage of neutrophils in BALF . ECP levels rose significantly in the DEHP+OVA group (p<0.05) in comparsion to the OVA group  between the DEHP+OVA group and OVA group. This case indicated that 10 mg/kg bw/day DEHP co-administration promoted AHR in OVA-sensitized mice, but pre-administration of anti-TSLP mAb effectively nullified this adjuvant effect, so values of Ri, Re and Cldyn in anti-TSLP group all changed significantly (p<0.05 or p<0.01), compared with that in DEHP+OVA group.To assess the change in AHR, we compared airway responses to MCH in different groups . In all As shown in p<0.01) in comparison to that in the saline group (p<0.05). Neutralization by anti-TSLP mAb significantly decreased PAS-positive cell count in the airway, when compared with that seen in the DEHP+OVA group (p<0.01)  .p<0.01) (p<0.05). Anti-TSLP mAb pre-administration decreased the degree of the collagen layer under the lamina propria in comparison to the DEHP+OVA group (p<0.05)  stained lung tissues . The colp<0.01) . The mor(p<0.05) .Several key findings were illustrated in the histological analysis of lung tissue slices stained with H&E, PAS or MT : (1) Thep<0.01 or p<0.05). The significant elevated TSLP levels in the DEHP+OVA group compared to the OVA group was one of the effects formed by DEHP co-exposure with OVA sensitization, which was so-called DEHP-adjuvant effect. Moreover, the TSLP levels in lung and BALF were both lower in the anti-TSLP group than in the DEHP+OVA group (p<0.05 or p<0.01).As the data in In this study, we concentrated on the possible association between TSLP and the hypersensitive reaction in a DEHP oral exposure mouse asthma model, and tried to discover the mechanisms relating to these DEHP immunoadjuvant effects.Recent animal studies have demonstrated that DEHP may promote and aggravate allergic asthma by an adjuvant effect –30. PrevTSLP anchors at the epithelial cells and acts as a key switch to trigger Th2 responses in allergic asthma, usually primarily produced by the damaged epithelial cells . Diverseet al [The typical markers of allergic asthma are significantly elevated IgE and enhanced airway hyperresponsiveness –43. In tet al . In addiet al . AHR is et al . When AHet al . DEHP, aet al , 48. HowThe adaptive immunity for allergic asthma is initiated when the allergen interacts with airway epithelia, so the bronchial epithelial cells play a vital role in the early stages of allergic asthma. In the present mouse asthma model, the allergen OVA induced the epithelial cells to secrete TSLP, when there was DEHP co-exposure with OVA, the levels of TSLP in pulmonary tissues and BALF were significantly increased. Excessive TSLP activated more dendritic cells and thus facilitated the priming and recruitment of more Th2 cells to the lung, and more Th2 cytokines and more allergen specific IgE antibody and IgG1 were produced Figs  and 8. TNotably, there were several special designs in the present study. First, the OVA group was set as the positive control facilitating the detection of potential DEHP-adjuvant effects, and the DEHP+OVA group was used as another positive control for detecting the effect of the anti-TSLP mAb neutralization in comparison to the anti-TSLP group. Second, subcutaneous administration, intraperitoneal injection, and inhalation exposure are the more effective methods for inducing the adjuvant effect of DEHP than oral exposure . EspeciaTaken together, our study demonstrates that DEHP at a dose of 10 mg/kg bw/day has immunoadjuvant effects on airway inflammation induced by OVA allergen, and TSLP is an effective target site for suppressing the adjuvant effect of DEHP co-exposure.S1 Fig(JPG)Click here for additional data file.


Recent retreat of outlet glaciers in Wilkes Land, East Antarctica, is driven by changes in sea ice. The dynamics of ocean-terminating outlet glaciers are an important component of ice-sheet mass balance. Using satellite imagery for the past 40 years, we compile an approximately decadal record of outlet-glacier terminus position change around the entire East Antarctic Ice Sheet (EAIS) marine margin. We find that most outlet glaciers retreated during the period 1974–1990, before switching to advance in every drainage basin during the two most recent periods, 1990–2000 and 2000–2012. The only exception to this trend was in Wilkes Land, where the majority of glaciers (74%) retreated between 2000 and 2012. We hypothesize that this anomalous retreat is linked to a reduction in sea ice and associated impacts on ocean stratification, which increases the incursion of warm deep water toward glacier termini. Because Wilkes Land overlies a large marine basin, it raises the possibility of a future sea level contribution from this sector of East Antarctica. This trend continued into the most recent epoch (2000–2012), when 65% of glaciers advanced at a median rate of 17.9 m year−1.Our results show clear decadal-scale patterns of terminus position around the entire EAIS marine margin (table S1). During the 1974–1990 epoch, 65% of glaciers retreated at a median rate of 17.8 m year−1). Between Queen Mary Land and Kemp Land (DB8 to DB12), there was a less obvious change, with 53% of glaciers retreating . During the periods 1990–2000 and 2000–2012, however, outlet glaciers within every drainage basin showed a dominant signal of advance  switch from the 1990s, when 75% of glaciers in Wilkes Land advanced. This is the first demonstration that glacier terminus changes in the region are linked to recently observed trends in mass loss  between each epoch reveal clear spatial trends across Antarctica . In EastMore recently, there has been only a small change in the overall average number of sea-ice days per year between 2000 and 2012, compared to the 1990s . HoweverCoastal polynyas are important in driving sea-ice variability in Antarctica. They produce around 10% of total sea ice, despite representing only 1% of the maximum sea-ice area . We suggChanges in the amount of sea-ice production can alter the mixing of shelf waters, which can drive variability in the basal melt rate of glacier termini , Thematic Mapper (TM), and Enhanced Thematic Mapper Plus (ETM+) satellites to map the terminus position of 176 outlet glaciers along the coast of East Antarctica across four approximate time steps: 1974, 1990, 2000, and 2012, stretching from Queen Mary Land to the Ronne Ice Shelf. This extends the record of a previous study (www.metoffice.gov.uk/hadobs/en4/download-en4-0-2.html). The objective analysis data set covers the entire study period on a monthly basis and is available at a 1° × 1° spatial resolution, with data obtained from the WOD09, GTSPP, Argo, and ASBO collections (www.metoffice.gov.uk/hadobs/en4/download-en4-0-2.html).We use the EN4.0.2 subsurface ocean temperature objective analysis data set . The data set offers near-complete coverage of the Antarctic sea-ice zone on a daily basis since July 1987 and every 2 days before this stretching back to October 1978 at a spatial resolution of 25 km × 25 km. There are brief gaps in the data set in August 1982  and 1984 (13 to 23 August). These have been interpolated where missing pixels were present (see http://nsidc.org/data/nsidc-0079).We used the Bootstrap sea-ice concentrations derived from the Nimbus-7 satellite and Defense Meteorological Satellite Program (DMSP) satellites (In line with other studies .https://legacy.bas.ac.uk/met/READER/). In addition, mean monthly 0.25° ERA-Interim (http://apps.ecmwf.int/datasets/data/interim-full-moda/levtype=sfc/) from 1979 to 2012 were extracted from the Wilkes Land coastline. For the purpose of this study, we defined the Wilkes Land coastline as a shapefile consisting of the coastline of DB13 with a 25-km buffer.Monthly mean surface air temperature records from Casey station were extracted from the Scientific Committee on Antarctic Research (SCAR) Met-READER project (


MycRNA expression, we quantified the relative levels of RNA for DNA damage-induced (DDI) genes, such asBax,Bbc3andCdkn1a. The RNA ratios of DDI genes/Mycin the blood increased in a dose-dependent manner 4 h after whole-body irradiation at doses ranging from 0.1 to 0.5 Gy (air-kerma) of X-rays, regardless of whether the mice were in an active or resting state. The RNA ratios were significantly increased after 0.014 Gy (air-kerma) of single X-ray irradiation. The RNA ratios were directly proportional to the absorbed doses in water ranging from 0.1 to 0.5 Gy, based on gamma-irradiation from137Cs. Four hours after continuous irradiation with gamma-rays or by internal contamination with a beta-emitter, the increased RNA ratios resembled those following single irradiation. These findings indicate that the RNA status can be utilized as a biodosimetric tool to estimate low-dose radiation when focusing on undifferentiated cells in blood.Biodosimetry, the measurement of radiation damage in a biologic sample, is a reliable tool for increasing the accuracy of dose estimation. Although established chromosome analyses are suitable for estimating the absorbed dose after high-dose irradiation, biodosimetric methodology to measure damage following low-dose exposure is underdeveloped. RNA analysis of circulating blood containing radiation-sensitive cells is a candidate biodosimetry method. Here we quantified RNA from a small amount of blood isolated from mice following low-dose body irradiation (<0.5 Gy) aimed at developing biodosimetric tools for situations that are difficult to study in humans. By focusing on radiation-sensitive undifferentiated cells in the blood based on Serious accidents at massive nuclear power plants lead to low-dose irradiation of a large number of victims. Equivalent doses to the victims due to the accidents can be estimated physically using the radiation dose rate of the evacuation route. For more precise dose estimation, however, biodosimetric data using biologic samples isolated from the victims are desirable. Dicentric chromosome analysis of lymphocytes is the most practicable method  and is ex vivoirradiated human blood [Measurement of γH2ax foci in chromosomes can be used to determine radiation-induced damage from a wide-range of doses . Practin blood  and blon blood ,16. Rn blood ,17, bn blood ,19 ann blood , are alCdkn1a,Mdm2,BaxandBbc3(PUMA) per housekeeping gene, such as glyceraldehyde-3-phosphate dehydrogenase gene (Gapdh), are increased in blood cells in a dose-dependent manner. The radiation-dose dependency of these mRNAs ratios, however, varies ∼2-fold between daytime and nighttime [MycRNA ratios linearly increased following low-dose radiation from 0.1 to 0.5 Gy in undifferentiated cells in the circulating blood both during the day and night. Using the biodosimetric model, we compared the DDI gene/MycRNA ratios after both a single exposure and continuous exposure. Effects on the RNA status of the radioprotector aminothiol were also examined.Experimental animal models can be used to study various modes of body-radiation that are difficult to evaluate in humans. The experimental data using animals of various ages and inbred strains may give us clues to speculate differences in biodosimetric parameters by the age and race in human beings. In mice ,22, rMice were treated in accordance with the Guidelines for the Proper Conduct of Animal Experiments (Science Council of Japan), and the Institutional Animal Care and Use Committee approved the study. Male C3H/He inbred mice at 7 weeks of age  were acclimatized at 3–5 mice per cage for 2 weeks in an animal room maintained at 23°C and 55% humidity. Mice were housed on a 12 h:12 h light:dark schedule, with white light on at 10–50 lux during the day. In the nighttime handling of mice, procedures were performed under blue LED light of <0.4 lux. The radioprotector cysteamine HCl  and the anesthetic pentobarbital sodium salt  were injected subcutaneously.Fewer than 10 mice were simultaneously whole-body irradiated using a cylindrical irradiation container ,23. F137Cs, the mice were placed in a cylindrical irradiation container and irradiated in a field with an estimated absorbed dose to water (EDw) rate of 0.535 Gy/min. For continuous irradiation, mice reared in cages were irradiated in a field of 0.032 mGy/h (EDw) of137Cs gamma-rays. These EDw values were determined by ionizing chamber measurement using a phantom mouse and the air-kerma values of the field, as measured by the Department of Technical Support and Development at our institute. For internal contamination experiments,32P-NaH2PO4 in phosphate-buffered saline solution was subcutaneously injected at doses of 80 or 200 MBq/kg-body weight.For single exposure to gamma-rays from32P was homogenously dispersed and all the energy from the β-rays was absorbed in the mouse, the EDw was estimated using the formula:η= modified factor [32P is 695 keV (1.11 × 10−13J). When the coefficient ofηwas 1.00, injections of 200 MBq/kg or 80 MBq/kg provided an EDw of 0.080 or 0.032 Gy/h, respectively.Based on the assumption that factor . The meBax,Bbc3,Cdkn1aandMycwere 2–8 ng, 5–20 ng, 5–50 ng and 10–100 ng, respectively. As a reference, linear plasmid DNA corresponding to similar molar amounts of all the Q-PCR target nucleotide sequences at 10 zeptomoles per well were used in all the reactions. TableThe details for accurate quantification of blood RNA were described previously . In briSignificant differences were determined by Dunnett's test following one-way analysis of variance. For regression analyses to determine the correlation coefficient, regression line and confidence interval, macros  of Microsoft® Excel® software were used.The need to collect only a small amount of peripheral blood would be advantageous following an enormous radiation disaster. The levels of intracellular molecules that reflect radiation damage in the specimen vary during storage. When the specimen is treated immediately after collection, however, the levels of the intracellular molecules can be maintained in storage for a long time. For practical future application of the mouse model, we focused on RNA of mice exposed to low-dose irradiation using a small volume of peripheral blood from the tail. When using circulating blood as a sample, the concentration of white blood cells (WBCs) drastically changed during the day in accordance with the circadian rhythm Fig.A, upperCdkn1a,BaxandBbc3are reported to be indicators of radiation-induced damage [Cdkn1amRNA is a growth-arrest indicator, andBaxandBbc3mRNAs are expressed during early apoptosis. These DDI genes are expressed in radiation-sensitive undifferentiated cells, which are presumed to be scarce in circulating WBCs , which is expressed predominantly in undifferentiated cells  are shown in Fig.MycRNA ratios, regardless of whether mice are in an active or resting state. Mice exposed to low doses of X-rays  had significantly higher RNA ratios in their blood  of X-rays.The RNA ratios in circulating WBCs were used to study the effects of a radioprotectorThe DDI RNA ratios in circulating WBCs are expected to be a useful dosimetry tool for localized irradiation, because WBCs exposed in local tissue are presumed to be dispersed throughout the circulatory system before blood collection. To confirm this, approximately two-thirds of the mouse body was irradiated during abdominal irradiation in mice anesthetized with pentobarbital, which does not affect DDI gene expression Figs.B,3B. 137Cs in which the EDw was controlled. The profiles of these RNA ratios in collected blood following whole-body irradiation with gamma-rays  varies following whole-body irradiation among different X-ray generators, because the radiation quality (i.e. a combination of various wavelengths of electromagnetic waves) from X-rays varies among generators. Based on the assumption that DNA damage in WBCs correlates with the EDw of radiation, mice were irradiated with a gamma-ray irradiator ofays Fig.A were says Fig.B. The cThere was an interval between the increase in biologic indicators, such as the DDI RNA ratios, and irradiation. After a single irradiation for <2 min, the RNA ratios peaked at 3 h and were maintained for at least 5 h after exposure FigsA. Conti32P. Injection of32P-PO4at 200 MBq/kg or 80 MBq/kg gave an EDw of 0.080 Gy/h or 0.032 Gy/h, respectively, when the32P radionuclide was homogenously dispersed, as described in the Materials and Methods. We observed dose-dependent increases in all of the DDI RNA ratios in WBCs from the injected mice  numbers in a single cell per 1 Gy are estimated to be ∼21  or ∼35 Cdkn1a, the increased levels are not remarkable in humans [The linearity of the DDI RNA ratios and exposed dose declined after low-dose radiation below 100 mGy Fig.E. Becau humans ,9 com humans . For fuBax/MycandBbc3/MycRNA ratios (Fig.Cdkn1a/MycRNA ratio was increased by cysteamine without irradiation (Fig.Aminothiols, such as the radioprotector amifostine, neutralize reactive oxygen species generated by irradiation . PretreMycRNA ratios after a single irradiation (Fig.The increase in the DDI gene/ion Fig.A was prion Fig.C. Immedion Fig.B. The r32P-phosphate was injected into mice and the WBCs used for analysis of the DDI RNA ratios (Fig.Bax/MycRNA ratio fell in the predicted ranges of 0.640 or 0.256 Gy. TheBbc3/MycorCdkn1a/MycRNA ratios, however, were above or below the predicted range, respectively. The deviation from the predicted range may be due to the long 8-h continuous exposure in which subsequent biologic processes can be triggered by the expression ofCdkn1a,BaxandBbc3mRNAs. These results demonstrated that the DDI RNA ratios in WBCs can be used to predict exposure doses after continuous irradiation. Further quantitative studies are needed, however, to determine more precise dosimetry.As another continuous irradiation model,ios Fig.D. Injecios Fig.C, the Rios Fig.B obtainQuantitative sptudies of RNA ratios focusing on undifferentiated cells in circulating blood, as described above, could be used for several applications, including biodosimetric technology and molecular biologic studies, following various types of body irradiation. Because the activity of the animal does not affect the RNA ratios, this method can be applied to animals that are difficult to breed or handle, or to wild animals. Knowledge gained from this study could be applied to experiments using human specimens, and to blood isolated from patients in hospitals on bed rest or from first responders in disasters immediately after exposure.All the works in this paper were supported by National Institute of Radiological Sciences, funded by Ministry of Education, Culture, Sports, Science and Technology of Japanese Government. Funding to pay the Open Access publication charges for this article was provided by the National Institute of Radiological Sciences.Supplementary DataClick here for additional data file.


The Child-Turcotte-Pugh score (CTP) is the standard tool for hepatic reserve assessment in hepatocellular carcinoma (HCC). Recently, we reported that integrating plasma insulin-like growth factor-1 (IGF-1) level into the CTP score was associated with better patient risk stratification in two U.S. independent cohorts. Our current study aimed to validate the IGF-CTP score in patients who have different demographics and risk factors.We prospectively recruited 100 Egyptian patients and calculated their IGF-CTP score compared to CTP score. C-index was used to compare the prognostic significance of the two scoring systems. Finally, we compared our results with our U.S. cohorts published data.IGF-CTP score showed significant better patient stratification compared to CTP score in the international validation cohort. Among CTP class A patients, who usually considered for active treatment and clinical trial enrollment, 32.5% were reclassified as IGF-CTP class B with significantly shorter OS than patients reclassified as class A with hazard ratio [HR] = 6.15, 95% confidence interval [CI] = 2.18-17.37.IGF-CTP score showed significantly better patient stratification and survival prediction not only in the U.S. population but also in international validation population, who had different demographics and HCC risk factors. While several hepatocellular carcinoma (HCC) prognostic systems rely on the Child-Turcotte-Pugh (CTP) score to assess the underlying chronic liver disease (CLD) and to predict treatment outcome and overall survival (OS). The origThe CTP score is still the standard tool for assessing the degree of hepatic reserve and predicting OS among cirrhotic and HCC patients., 3, 4 HoOur previous studies, 14 showMD Anderson training cohort (USA): 420 HCC patients were enrolled between January 2000 and May 2008. Among those, 310 (73.8%) had available plasma samples.MD Anderson prospective validation cohort (USA): 197 HCC patients were enrolled between June 2008 and September 2011. Among those, 155 (78.7%) had available plasma samples.Assiut prospective validation cohort (Egypt): 100 HCC patients were enrolled between April 2012 and September 2013, and all had available plasma samples taken on the day of study enrollment in the clinic.The median follow-up times were 8.6 months (95% CI=6.8 to 14.5 months) for the Egyptian validation cohort, compared to 43.3 months (95% CI=41 to 53.5 months) for the training cohort and 16.5 months (95% CI=9.67 to 24.1 months) for the MD Anderson validation cohort.Table P < .0001); the same was true for both the training and MD Anderson validation cohorts (P < .001).The median OS duration in the Egyptian validation cohort was 8.05 months (95% CI=6.9 to 9.2 months), compared to 13.2 months (95% CI=11.4 to 16.6 months) in the training cohort and 15.7 months (95% CI=12.2 to 19.9 months) in the MD Anderson validation cohort. Table P < 0.0001), as well as in the training cohort (P = 0.001). In the MD Anderson validation cohort, patients with a low IGF-1 level had a worse prognosis than that of patients with an intermediate IGF-1 level, but this difference was not statistically significant .Among all three cohorts, patients with low IGF-1 levels had statistically significantly shorter OS compared to patients with high IGF-1 levels. Patients with an intermediate IGF-1 level had shorter OS compared to patients with a high IGF-1 level, but this difference was not statistically significant in either validation cohort. Furthermore, patients with a low IGF-1 level had shorter OS compared to patients with an intermediate IGF-1 level in the Egyptian validation cohort (P = 0.003) as well as in the training cohort (P = 0.003) and MD Anderson validation cohort (P = 0.005). Similarly, MELD score study reported a C-index of 0.83 for MELD score as compared to 0.76 for the CTP. Notably, the difference between C-indices was not large (=0.07), however, it was statistically significant, P < 0.001. This is because the C-index computes the prognostic score ability to predict OS for all patients in the cohort, including those whose CTP and IGF-CTP scores were different and those whose scores were the same. To better understand this improvement, we focused on the patients who were classified into different risk groups by the two scoring systems.Table We found that 63% of patients in the Egyptian cohort were classified in the same risk groups by both scoring systems, compared to 61.9% in the training cohort and 53.5% in the MD Anderson validation cohort Figure  showed aP = 0.001 and HR=10.11; P = .03, respectively).In the Egyptian validation cohort, 26/40 (65%) of the patients with CTP class A were classified as IGF-CTP-A (AA), and they had an OS duration of 12.58 months (95% CI=11.7 to 13.4 months). While, 13/40 (32.5%) were reclassified as IGF-CTP-B (AB), and they had significantly worse OS of 8.9 months (95% CI=7.8 to 10 months) compared to group AA. Only one patient was reclassified as IGF-CTP-C (AC). Patients reclassified from CTP-A to IGF-CTP-B (AB) or IGF-CTP-C (AC) had worse prognosis compared to patients who were classified as AA . Finally, the majority of CTP-C patients remained in class C according to the IGF-CTP scoring system. Only 7/27 (25.9%) were reclassified as IGF-CTP-B, and their OS duration was 8.05 months (95% CI=6.9 to 9.2 months); this duration did not differ significantly when compared to patients who were classified as CC .Moreover, patients who were reclassified from CTP-B to IGF-CTP-A (BA) had better prognosis compared to patients who were classified as BB , and BCLC. Several studies have concluded that only patients with CTP class A are likely to benefit from active treatment and enrollment into clinical trials, while patients with CTP class B or C disease have significantly decreased survival expectancy due to the deterioration in hepatic function compared to class A., 4 When The major strength of our study is that this is the first international validation of the IGF-CTP score, and it is tested through an independent prospective validation cohort in patients with different demographics, geographical location, hepatitis status and other HCC risk factors from those of the original cohorts. In the Egyptian cohort, all patients had viral-induced HCC, compared with only 44.8% in the training cohort and 50.3% in the first validation cohort. Our findings help to identify the prognostic significance of this score in patients who have more advanced disease and marked impairment of the liver capacity both by HCC as a space-occupying lesion and by the underlying viral hepatitis, which is mainly induced by HCV genotype 4, with a higher degree of liver fibrosis. Another strength of this study is that since the majority of patient's had unresectable disease; the main pool of patients undergoing systemic and local therapies in routine practice and clinical trials; accurate assessment of hepatic reserve and subsequent prediction of patient's prognosis and survival in this group of patients is very important to identify who will get benefit from active therapy and potential enrollment in clinical trials.“Our study has some limitations. First, the majority of patients had unresectable disease. However, predicting patient's prognosis and survival in this group of patients is very important to identify who will benefit from systemic therapy and potential enrollment in clinical trials. Second, we don't have complete data regarding alcohol intake. However, all cases were viral related and therefore cirrhosis in the Egyptian population was mainly related to hepatitis virus. Finally, A limitation of our study was that it didn't allow us to test the predictive value of the IGF-CTP score for the outcome of different treatment modalities. Therefore, future studies to evaluate the predictive ability of IGF score in systemic and local therapies are warranted”.Despite the CTP score's limitations, it is still the only standard tool parameter for assessing underlying liver reserve capacity within the most clinically used staging systems as CLIP, CUPI, and BCLC. So, this will affect patient staging and subsequantly selecting the suitable treatment, predicting treatment outcome, and identifing patients who are eligible to be enrolled in clinical trials. In the current study, we validate the value of incorporating plasma IGF-1 level instead of encephalopathy and ascites as parameters of the CTP score to create a new scoring system, Kaseb-Morris IGF-CTP system.The new IGF-CTP score significantly improved selection of patients who were candidates for treatment and prediction of survival outcome. Since the majority of HCC staging systems include CTP score in their parameters,1 future studies to assess the value of integrating IGF-CTP score instead of CTP score in these staging systems may lead to more accurate patient stratification and treatment selection.” After further validation in HCC patients with different demographics and risk factors, the new IGF-CTP score may help to improve prediction of survival outcomes and rate of therapy adverse events which may subsequently aid in better selection of patients who are candidates for active HCC treatment and in prediction of survival outcome. Finally, since the majority of HCC staging systems include CTP score in their parameters as a liver assessment tool,1 future studies to assess the value of integrating IGF-CTP score instead of CTP score in these staging systems may lead to more accurate patient stratification and treatment selection.We recruited 100 HCC patients from Assiut University Hospital, Egypt, from April 2012 to September 2013. We compared our cohort with two previous cohorts: i) prospectively recruited HCC patients who presented at MD Anderson Cancer Center from January 2000 to May 2008 and were used as a training cohort, and ii) prospectively recruited HCC patients who presented at MD Anderson between June 2008 and September 2011 and were used as a validation cohort. This study was approved by the institutional review boards of MD Anderson and Assiut University Hospital. Written informed consent for study participation was obtained from each patient. For all three HCC cohorts, the study involved pathologically confirmed HCC or radiologically confirmed HCC based on American Association for the Study of Liver Diseases guidelines. for patiDetailed data on patients’ demographics, risk factors, and clinicopathological features were collected. The clinical data included information about Eastern Cooperative Oncology Group (ECOG) performance status; levels of alpha-fetoprotein (α-FP), aspartate transaminase (AST), and alanine transaminase  stage; and treatment history. We followed standard approach to calculate CTP score, by using clinical judgment and imaging studies to assess ascites and encephalopathy which is currently the standard approach in routine practice and clinical trials, therefore limited since it is subjective parameters and influenced by other factorsBlood samples for IGF-1 analysis were prospectively collected and analyzed for IGF-1 in our cohort. Our data were compared with the IGF-1 values in the training cohort, for which samples were prospectively collected and retrospectively analyzed, and in the validation cohort, for which samples were prospectively collected and analyzed.Peripheral venous blood samples (3-5 mL) were collected, anticoagulated by ethylenediaminetetraacetic acid (EDTA), and centrifuged at 4°C for 15 minutes at 3000 RPM. Then the plasma samples were removed, aliquoted, and snap-frozen at −20°C until analyzed.In both the training cohort and the Egyptian validation cohort, IGF-1 was tested by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's directions . In the MD Anderson validation cohort, plasma IGF-1 was tested at a Clinical Laboratory Improvement Amendments (CLIA)–certified facility that uses Luminex microsphere technology by Myriad Laboratories .Patients’ IGF-CTP scores were calculated and class A, B, or C assigned based on their serum bilirubin level, serum albumin level, prothrombin time, and plasma IGF-1 level as described previously.P value as a descriptive measure was considered statistically significant if less than 0.05.We used IBM SPSS Statistics for Windows, Version 21.0  for data management and statistical analysis. Univariate analysis was done using chi-square or Fisher's exact test for categorical variables and the Kruskal-Wallis test for continuous variables. The Kaplan-Meier method was used to estimate the median overall survival (OS), which was defined as the time interval between the date of the blood draw for IGF-1 measurement and the date of death or last follow-up. Also, the log-rank test was used to detect the statistical significance between the CTP score and the new IGF-CTP score subgroups. Then, we used the Cox proportional hazard model to calculate hazard ratios (HRs) and 95% confidence intervals (95% CIs) to identify independent prognostic factors for OS. Finally, we calculated the Harrell's C-index to compare the prognostic performance of both scores among the three cohorts. For all statistical analyses, a two-sided


The transfected BMSCs were then inoculated onto the scaffold material A-W-MGC/CS to construct tissue-engineered bone. The attachment and proliferation of BMSCs were observed by scanning electron microscopy (SEM) and 3--2,5-diphenyltetrazolium bromide (MTT) detection, respectively. Rabbit models of bone defects were established and divided into three groups. Experimental group 1 was implanted with prepared tissue-engineered bone. Experimental group 2 was implanted with A-W-MGC/CS without transfected BMSCs. The blank group was injected with transfected BMSCs, without implantation of any scaffold. In the 12th week after surgery, the repair of bone defect was observed by X-ray examination, and histological observations of the area of bone defect were performed.A-W-MGC/CS resulted in good BMSC attachment and had no obvious effects on cell proliferation. In experimental group 1, good repair of bone defect was observed, and the scaffold material degraded completely. In experimental group 2, new bone was formed, but its quality was poor. In the blank group, there was mainly filling of fibrous connective tissues with no observable bone defect repair.in vivo osteogenic capability for bone defect repair.A-W-MGC/CS possesses good biocompatibility and  Autogenous bone grafting is the most commonly used method, but the bone supply is limited and some complications may occur in the donor area ,2. As alThis study was conducted with approval from the Animal Ethics Committee of Liaoning Medical University. Bone marrow (4 mL) was drawn from the proximal tibia of a Japanese white rabbit  and was added to a sterile flask. Then 4 mL of DMEM containing 10% fetal bovine serum (FBS) was added to the flask . After repetitive beating, 2 mL of the mixture was transferred to another flask, followed by the addition of 2 mL of culture medium. After 5 days, the culture medium was replaced with fresh medium. The growth of bone marrow stromal cells (BMSCs) was observed. When 80% of adherent cells were fused, the primary culture was closed and subculture was performed. The third generation of cells was collected for use.5 cells/bottle) was transfected by adenovirus-human bone morphogenetic protein-2-green fluorescent protein (Ad-hBMP2-GFP) (previously prepared in our laboratory) with a multiplicity of infection (MOI) of 100. The transfection results were observed at 24 h and 48 h under a fluorescence microscope. Flow cytometry  was performed for cell counting. The results were expressed as transfection rate and mean fluorescence intensity of GFP-positive cells.The third generation of BMSCs (5 × 106 cells/mL) was inoculated onto A-W-MGC/CS to prepare the tissue-engineered bone, followed by transfer into a 24-well plate for incubation. After 2 h, the tissue-engineered bone was turned over. After 4 h, 1.5 mL of DMEM containing 20% FBS was added to each well for 3 days of culture. The culture medium was replaced with fresh medium once a day.A-W-MGC/CS  was disinfected using ethylene oxide. Before use, A-W-MGC/CS was washed three times with PBS and then immersed in DMEM without FBS for 7 days. Finally, the culture medium was removed and A-W-MGC/CS was naturally dried in an incubator. A suspension of transfected BMSCs  were enrolled in the establishment of bone defect models. A longitudinal incision (length of 3 cm) was made in the anteriomedialis of the left and right forearms. The skin and fascia were incised, followed by layer-by-layer separation of muscle, vessel, and nerve. The middle radius was fully exposed. A 1.5-cm bone defect was made, and the periosteum in the defect area was carefully removed. The rabbit models of bone defect were established and divided into three groups. After washing the incision with normal saline, experimental group 1 was implanted with A-W-MGC/CS with transfected BMSCs. Experimental group 2 was implanted with A-W-MGC/CS without transfected BMSCs. The blank group was injected with transfected BMSCs without implantation of any scaffold. Finally, the incision was closed layer-by-layer. Each rabbit was reared in a separate cage without intraoperative and postoperative fixation.The third generation of BMSCs was inoculated in a 96-well plate at a density of 2,000 cells/well. Culture medium (200 μL) immersing A-W-MGC/CS was added to 21 wells , and 200 μL of normal culture medium was added to another 21 wells (control group). The medium was replaced with new medium once every 48 h. Each day, three wells of cells were taken and 20 μL of 3--2,5-diphenyltetrazolium bromide (MTT) was added to each well. After 4 h, the culture solution in the wells was aspirated. Then 150 μL of DMSO was added, followed by oscillation for 10 min. The optical density of the mixture was measured at a wavelength of 490 nm. A blank well was used as a control.BMSCs were inoculated into a 96-well plate at a density of 2,000 cells/well. In the experimental groups, after cell adherence, BMSCs were transfected by Ad-hBMP2-GFP (MOI = 100), followed by routine culture. In the control group, BMSCs were not transfected by Ad-hBMP2-GFP. On the 3rd, 6th, 9th, and 12th day, the supernatant of each well was removed and the wells were washed with PBS. Then 100 μL of 0.1% Triton X-100 was added to each well for overnight cleavage (4°C). The alkaline phosphatase (ALP) activity was determined using ALP kits  according to the manufacturer’s instructions. Using continuous monitoring methods (3 min), the absorbance change per minute (ΔA/min) was detected at a wavelength of 405 nm. The actual concentration of ALP (U/L) was calculated according to the following formula: U/L = ΔA/min × 2,757.After 3 days in culture, the cell-and-scaffold-composite specimen was taken out. After washing three times with PBS, 2.5% glutaraldehyde was used for fixation at room temperature, followed by graded dehydration with 60%–100% ethanol and replacement with isoamyl acetate. Then the specimen was dried at critical point temperature, followed by surface coating with gold and scanning electron microscope (SEM) observation.Nude mice were bought from the laboratory of the animal center at the Chinese Medical Sciences University . The left side of the femoral muscle was used as the experimental side (Ad-GFP-hBMP was added), and the right side was used as the control side . Animal responses were generally observed after transfection into the femoral muscles of nude mice. X-ray films were taken after 12 weeks to observe the ectopic osteogenesis effect. Then these mice were killed and samples were obtained for hematoxylin and eosin (HE) staining.At the 4th, 8th, and 12th week after surgery, X-ray examination  was conducted on the bilateral ulna and radius of the rabbits.At the 4th, 8th, and 12th week after surgery, six rabbits were executed. The ulna and radius in the area of bone defect were taken. The degradation of the implanted material, the combination with the host bone interface, and the reflection of the surrounding soft tissues were observed. The specimen was then treated by decalcification with 5% nitric acid for 72 h, followed by fixation with 4% formalin. After embedding in paraffin, slices with 10-μm thickness were continuously prepared and subjected to HE staining and observation.s. Statistical analysis was performed using SPSS 17.0 statistical software. Single factor ANOVA was conducted for comparison between different groups. P < 0.05 was considered as statistically significant.Data were expressed as P > 0.05), suggesting that the medium immersing A-W-MGC/CS did not affect the growth and proliferation of BMSCs . The ALP levels at each time point in the experimental group were higher than those in the control group. This result indicated that the transfected BMSCs could be transplanted into the body to promote osteogenesis in the bone defect area.Table An image of the A-W-MGC/CS scaffold is shown in Figure In the experimental group, X-ray films suggested that seven sides exhibited a high-density shadow, two sides exhibited a slightly high-density shadow, and one side did not show anything. The positive rate of ossification was 90%. In the control group, four sides exhibited a high-density shadow with uneven density, while six sides did not show anything, and the positive rate of ossification was 40% Figure A. HistolAt the 4th week after surgery, in experimental group 1, continuous bone callus appeared at the center of the implanted material, and bilateral bony callus uniformly extended to the middle. The interface between the host bone and the tissue-engineered bone was fuzzy. In experimental group 2, there was continuous bone callus with a density lower than that of normal bone. In the blank group, no obvious bone callus was visible in the bone defect area Figure . At the The results of histological observation at the 4th, 8th, and 12th week after surgery are shown in Figure in vivo tracing [vitro. According to previous experimental results [As seed cells, BMSCs have stable characteristics and can be easily obtained and amplified. In addition, they have the potential of multi-directional differentiation and have been widely used in tissue engineering. The cytokine BMP2 can promote bone defect repair and fracture healing, thus exhibiting an osteoinductive activity -12. The  tracing -15. As a tracing . In this results , the MOIin vivo dissolution of AW-MGC/CS could increase the concentration of Ca2+, thereby enhancing the gene expression of transforming growth factor-β1, improving the activities of osteoblasts, and promoting their differentiation. Meanwhile, the osteoblasts could directly calcify on the bone surface. The osteoid among the surface and among cells would directly calcify, and did not form layers of connective tissue membranes. The degradation rate of AW-MGC/CS was lower than that of β-TCP, thus avoiding the phenomenon of excessive degradation-induced “powder residue stasis,” and the Ca-P-rich layer generated during this degradation could be directly used by the osteoblasts to form bones.Ideal extracellular matrix materials in bone tissue engineering should satisfy the following requirements -20. (1) In vitro SEM observations showed that the cells had good adherence and growth with uniform distribution, regular form, and good attachment to the surface of the implanted material. These observations confirmed the feasibility of A-W-MGC/CS as a carrier of seed cells for bone tissue engineering. In vivo experiments also proved that A-W-MGC/CS possessed a high ability of ossification and bone defect repair. (2) Excellent biodegradability. Histological observations showed that at the 4th week after implantation, in experimental group 1, the implanted material has degraded into a mesh-shaped structure, and the newly formed osteoid tissues perforated in it. Cartilage-like cell clusters were distributed in the implanted material. In experimental group 2, many chondrocytes were found in the area near the ulna and the fractured end, which may be related to the participation of osteoblasts at the ulnar periosteum and the fractured end in bone repair. At the 12th week, the implanted material has virtually degraded completely. (3) Porousness and high porosity. The carrier had a three-dimensional porous structure, and the pores were connected to each other with uniform distribution. The aperture was 100–500 μm with a porosity of 80%–90%. In this study, SEM examinations showed good adherence and growth of cells on the implanted material. Histological observations showed that in experimental group 1, the new bone was uniformly distributed on the transverse section of the bone defect area, suggesting that the growth of new bone was uniform. After implantation of the tissue engineered bone, the seed cells on the implanted material not only proliferated well and secreted more extracellular matrix, but also retained the basic biological characteristics. These cells absorbed nutrients from the blood and extracellular fluid in the area of surgery. After a period of time, the surrounding capillaries grew into the pores in the implanted material and exchanged nutrients and metabolites with the seed cells. As a result, the cells could continue to proliferate and secrete large amounts of extracellular matrix. Histological observations showed that osteoid tissues were present in the implanted material with newly formed vessels. Observations of tissue sections found that the growth of new bone on the transverse section of the bone defect area was uniform with no decrease in osteogenic ability due to cell death in the implanted material. (4) Plasticity and a certain degree of mechanical strength. The scaffolding material could be molded into a variety of complex configurations according to the foam body shape. The mechanical strength of the scaffold has been validated in early experiments, with no appearance of second fracture in in vivo experiments. (5) Osteoconductivity and osteoinductivity. In this study, continuous bone callus appeared early in the bone defect area. Histological observations showed that osteoid tissues were also present in the central bone defect area. MTT results found that A-W-MGC/CS had good biocompatibility, no cytotoxicity, and did not affect the growth and proliferation of cells. In addition, during the whole stage after implantation of the scaffolding material, no signs of acute inflammation were detected in the bone defect area except for the infiltration of a small number of lymphocytes at the 4th week. This observation indicated that the scaffolding material exhibited high biocompatibility without causing immune rejection to the body.The AW-MGC/CS-constructed tissue-engineered bone, combined with Ad-GFP-hBMP-transfected BMSCs, exhibited better osteogenic repairing abilities towards bone defects. These results indicated that the scaffold applied to treat bone defects could affect osteoconductivity.


Non-alcoholic fatty liver disease (NAFLD) caused by liver lipid dysregulation is linked to obesity. Somatostatin (SST) and its analogs have been used to treat pediatric hypothalamic obesity. However, the application of such drugs for the treatment of NAFLD has not been evaluated.This study aimed to investigate the expression levels of important regulators of hepatic lipid metabolism and the possible effect of the SST analog octreotide on these regulators.SD rats were assigned to a control group and a high-fat diet group. Obese rats from the high-fat diet group were further divided into the obese and octreotide-treated groups. The body weight, plasma SST, fasting plasma glucose (FPG), insulin, triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and free fatty acid (FFA) levels were measured. Hepatic steatosis was evaluated based on the liver TG content, HE staining and oil red O staining. The SREBP-1c, ACC1, FAS, MTP, apoB and ADRP expression levels in the liver were also determined by RT-PCR, qRT-PCR, western blot or ELISA.The obese rats induced by high-fat diet expressed more SREBP-1c, FAS and ADRP but less MTP protein in the liver than those of control rats, whereas octreotide intervention reversed these changes and increased the level of apoB protein. Compared to the control group, obese rats showed increased liver ACC1, SREBP-1c and apoB mRNA levels, whereas octreotide-treated rats showed decreased mRNA levels of apoB and SREBP-1c. This was accompanied by increased body weight, liver TG contents, FPG, TG, TC, LDL-C, FFA, insulin and derived homeostatic model assessment (HOMA) values. Octreotide intervention significantly decreased these parameters. Compared to the control group, the obese group showed a decreasing trend on plasma SST levels, which were significantly increased by the octreotide intervention.Octreotide can ameliorate hepatic steatosis in obese rats, possibly by decreasing hepatic lipogenesis and increasing TG export from hepatocytes. At the end of week 24, 32 eligible obese rats with a mean body weight at least 1.4 times heavier than that of the control group were selected from the high-fat diet rats [Fifty-six healthy, 21-day-old male Sprague-Dawley rats were purchased from the Animal Center of Sichuan University. All rats were housed in individual cages with 12 h light-dark cycles and given free access to water and chow. After 3 days of adjustable feeding at the Animal Center of West China Hospital, the rats were randomly divided into two groups as follows: the first group containing 16 rats was maintained on standard chow  for 176 days which was set as the normal control group, whereas the second group containing 40 rats was fed high-fat chow . It has reported that such high-fat diet formulation induces obesity in rats [iet rats . On the iet rats , 18. FooThe rats were fasted for 12 hours and anesthetized with 2% sodium pentobarbital administered intraperitoneally at the end of the experiments. The liver and abdominal adipose  were collected and weighed. Portions of the liver were either frozen at -80°Cfor the analysis of gene and protein expression or fixed in 4% paraformaldehyde for further histopathological analysis. The blood samples were centrifuged  to separate the plasma and then stored at -80°C for use.The Institutional Animal Care and Ethic Committee of Sichuan University (Approved No. SYXK 2008–119) approved all animal studies.125I-SST with its antibody. Therefore, the total binding to SST antibody in the plasma after octreotide injection was defined as SST-like immunoreactivity (SST-LIR) [The fasting plasma glucose (FPG) concentration was determined with a blood glucose assay . The fasting serum insulin was determined with a radioimmunoassay kit . To estimate the insulin sensitivity, the homeostatic model assessment (HOMA) value was calculated as previously described . The serSST-LIR) .2 and subjected to frozen sectioning. Then, the frozen tissue sections (9-μm thickness) were immersed in 1% oil red O working solution for 10 min, counterstained with hematoxylin, and then rinsed under running tap water for 30 min. Photomicrographs were captured under a microscope  from different views. The integrated optical density (IOD) of the oil red O-stained area was analyzed with Image-Pro Plus 6.0 software .Liver tissues were fixed, dehydrated and embedded in paraffin. Paraffin sections (3-μm thickness) were then subjected to hematoxylin and eosin (HE) staining. The liver tissue morphology and unstained areas of fat vacuoles were observed under a light microscope. Oil red O (ORO) staining was performed to detect neutral lipids and lipid droplet morphology with slight modifications . BrieflyTo determine the liver lipid content, hepatic lipids were extracted . BrieflyIn a separate experiment, the rats from three groups  were fed as described above. At the end of the octreotide treatment, three groups of rats were fasted for 12 h and injected with Triton WR-1339  via the tail vein at a dose of 500 mg/kg body weight ,24. PlasA linear relationship was observed between VLDL-TG concentrations and time after i.v. injection of Triton WR-1339, and the VLDL-TG secretion rate was calculated as the slope of this linear curve .The total RNA from 10 mg of liver tissue was isolated using Trizol reagent  following the manufacturer’s instructions. The concentration and purity of the RNA samples were then measured. The RNA was reverse-transcribed into cDNA by using reverse transcription kits  and then stored at -80°C. The cDNA template was then used to analyze the MTP, ACC1 and apoB mRNA levels via polymerase chain reaction (PCR). Intron-spanning primers were synthesized by the GeneWeiZ biotechnology company of Suzhou China and were listed in TM5 Real Time PCR Detection System. The expression of ADRP, FAS, and SREBP-1c was analyzed from the real-time PCR assay, which was normalized using the 2-ΔΔCtCt method relative to β-actin.The 2×SYBR Green qPCR Master Mix qRT-PCR RNA Detection Kit  was used in conjunction with SYBR Green for the quantification of the ADRP, FAS, and SREBP-1c genes. Total RNAs from the liver tissues were extracted using Trizol reagent . The cDNA was obtained from reverse transcription (RT) reactions. Briefly, ~50 ng of total RNA was subjected to reverse transcription with random primers according to the manufacturer’s instructions. Then, the cDNA products were used for SYBR green-quantitative real-time PCR reaction together with the relative primers . The ampThe concentration of the proteins extracted from the livers was quantitated with a BCA protein assay kit  prior to mixing with loading buffer and heating at 100°C for 5 min. The extracted protein (40 μg) was separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes . Nonspecific binding sites were blocked with 5% non-fat dry powdered milk in incubation buffer, and the membranes were then incubated with antibodies against rat MTP , SREBP-1c , or ADRP  at 4°C overnight. The membranes were washed, followed by incubation with the appropriate HRP-conjugated secondary antibodies for 2 h at room temperature . Specific bands were visualized by ECL detection. The band densities were quantified using Quantity One software 4.6.2. The housekeeping protein β-actin or GAPDH was analyzed for normalization.2O2 for 10 min. After blocking with goat serum, the sections were incubated with anti-FAS  and anti-ADRP (1:500) overnight at 4°C. The FAS and ADRP proteins were detected by incubating the sections with biotinylated secondary antibodies to form streptavidin-biotin-complexes. Finally, the immunocomplex was visualized by a solution of diaminobenzidine tetrahydrochloride, and the sections were counterstained with hematoxylin. Three fields were randomly selected for each rat liver, and the IOD value was analyzed with Image Pro Plus 6.0 software.After deparaffinization, sections of liver tissue were subjected to high-pressure antigen retrieval for 10 min in citrate buffer at pH 6.0 and treated with 3% HIn brief, liver tissue (40 mg) was homogenized at 4°C in phosphate buffered saline (pH 7.4) and centrifuged at 2000 rpm for 20 min at 4°C. The levels of apoB protein were determined using an ELISA kit  following the manufacturer’s instructions.Data are presented as the mean± SD and were analyzed with SPSS 16.0 statistical software . Comparisons among three groups were performed by one-way ANOVA test, and then Student-Newman-Keuls test was applied to detect significant differences. A test for the homogeneity of variances was also necessary. Differences were considered statistically significant when P values were less than 0.05.th week, final body weight, body weight gain and Lee’s index were significantly greater in the obese rats compared to these of the control group (p<0.01). The final body weight and body weight gain of the octreotide-treated group were significantly decreased compared to the obese group (p<0.05). However, Lee’s index did not differ significantly between these two groups. In addition, octreotide was effective in attenuating the increased abdominal fat and fat/body weight ratio in obese rats.Body weight gain was calculated by the difference between final body weight at the end of the experiments and original body weight in all rats within the 176-day period. As shown in p<0.01). The food/energy intake on the HFD was not significantly different between the obese group and the octreotide-treated group. The octreotide intervention had no effect on food/energy intake in the obese rats.The consumption of a high-fat diet led to a decrease in food intake (g/day/rat); however, energy intake  in the obese group was 27.3% higher than that in the control group (p<0.05). As the good cholesterol, the HDL-C concentration was lower in the obese group compared to that in the control group (p<0.05); however, octreotide was not effective in increasing the HDL-C levels. Compared to the control group, the plasma SST levels in the obese group were decreased, but there was no significant difference (p>0.05). Nevertheless, the plasma SST levels notably increased in the octreotide-treated group compared to the obese group (p<0.05).As shown in p<0.01; p<0.01). Consistent with these results, the TG content in the liver tissue was 3.2 times higher in the obese rats than in the normal control group, whereas the levels of liver TG decreased by 35.5% after octreotide treatment (It can be seen from reatment .p<0.01), but octreotide treatment induced decreases of immature and mature SREBP-1c proteins by approximately 69.3 and 48.8% (p<0.01), respectively. The results of the immunohistochemical staining showed that the up-regulation of FAS in the livers of the obese group significantly decreased in response to octreotide treatment (p<0.05). Unexpectedly, the liver ACC1 mRNA levels in the obese and octreotide-treated groups showed no difference . To our best knowledge, the VLDL-TG secretion rate was calculated as the slope of the curve linearization between VLDL-TG concentrations and time [p<0.01; p<0.01; and time . The VLDp>0.05; p<0.01; The MTP mRNA levels showed no difference among the three groups p>0.05; ; howeverp<0.01; p>0.05). However, the apoB protein level in the octreotide-treated group was significantly increased by 27% compared to the obese group .The immunohistochemistry indicated that the ADRP protein localized to the surface of the lipid droplets (LDs) . The immA high-fat diet tends to induce obesity, which increases the prevalence of liver steatosis . In our Liver steatosis arises from an imbalance between lipid input (fatty acid uptake and de novo lipogenesis) and output (VLDL export and fatty acid oxidation) . SREBP-1VLDL is the principal vehicle for the export of endogenous TG in the liver. The assembly and maturation of VLDL are hypothesized to occur as two steps. The first step is the lipidation of apolipoprotein B (apoB) with a few lipids aided by MTP, forming lipid-poor pre-VLDL particles , 33. TheApoB is the main structural protein of VLDL. In this study, the expression of apoB mRNA was increased in obese rats compared to the control group, but the expression of the apoB protein did not differ between these two groups. Previous studies of heterozygous MTP knockout mice suggested that half-normal levels of MTP decrease the level of plasma apoB100 to the same extent (25–35%) at any level of apoB synthesis . TherefoThe esterification and maturation processes of VLDL are associated with ADRP. The level of TG in the cytosol is reduced in ADRP-deficient mice, whereas TG concomitantly accumulates in the microsome . The micIn fact, octreotide has been used to cure clinical diseases for several decades, such as pancreatitis and gastrointestinal bleeding, with relative safety including no toxicity to the liver and kidney and no genetic-related side effects. As a bioactive molecule, however, many other functions of octreotide were overlooked. In this study, we found that octreotide improved nonalcoholic fatty liver disease, suggesting that octreotide is a novel promising drug for treating fatty liver disease.To our knowledge, unsatisfactory safety has limited the practical use of conventional drugs for nonalcoholic fatty liver disease. Unlike conventional drugs, octreotide is an analog of endogenous somatostatin (SST). In addition, octreotide can improve the low level of somatostatin in obesity, improve insulin resistance and pancreatic fat, and inhibit the intestinal absorption of fat. These properties indicate the good biocompatibility of octreotide. Here, we have shown that octreotide decreases rat liver fat levels by regulating SREBP-1c, ACC, FAS, MTP, apoB, and ADRP expression. However, the detailed molecular mechanism remains uncertain. It was reported that the adenosine monophosphate-activated protein kinase (AMPK) pathway plays an important role in modulating hepatic lipogenesis , 48, 49.S1 File(DOC)Click here for additional data file.S2 File(DOC)Click here for additional data file.S3 File(DOC)Click here for additional data file.S4 File(DOC)Click here for additional data file.S5 File(DOC)Click here for additional data file.S6 File(DOC)Click here for additional data file.


Klebsiella pneumoniae is an opportunistic pathogen causing nosocomial infections. Main virulence determinants of K. pneumoniae are pili, capsular polysaccharide, lipopolysaccharide, and siderophores. The histone-like nucleoid-structuring protein (H-NS) is a pleiotropic regulator found in several gram-negative pathogens. It has functions both as an architectural component of the nucleoid and as a global regulator of gene expression. We generated a Δhns mutant and evaluated the role of the H-NS nucleoid protein on the virulence features of K. pneumoniae. A Δhns mutant down-regulated the mrkA pilin gene and biofilm formation was affected. In contrast, capsule expression was derepressed in the absence of H-NS conferring a hypermucoviscous phenotype. Moreover, H-NS deficiency affected the K. pneumoniae adherence to epithelial cells such as A549 and HeLa cells. In infection experiments using RAW264.7 and THP-1 differentiated macrophages, the Δhns mutant was less phagocytized than the wild-type strain. This phenotype was likely due to the low adherence to these phagocytic cells. Taken together, our data indicate that H-NS nucleoid protein is a crucial regulator of both T3P and CPS of K. pneumoniae. Klebsiella pneumoniae is an opportunistic Gram-negative bacterium belonging to the Enterobacteriaceae family causing nosocomial infections such as septicemia, pneumonia, urinary tract infections, surgical site infections and catheter-related infections , lipopolysaccharide, siderophores, and pili , Type 3 pili (T3P), and E. coli common pilus  is a DNA-binding protein found in enteropathogens such as K. pneumoniae. The absence of H-NS down-regulated the T3P and affected biofilm formation. In contrast, expression of CPS was derepressed in a Δhns mutant, conferring a hypermucoviscous phenotype. Finally, the absence of H-NS resulted in low adherence to epithelial cells and macrophages and in high resistance to macrophage phagocytosis.In this work we describe the effect of H-NS protein on the expression of both T3P and CPS, two of the main virulence determinants of Bacterial strains and plasmids used in this study are listed in Table K. pneumoniae 123/01 was isolated from a patient with pneumonia by bronchoalveolar washing. Capsular serotype K39 was determined by sequencing of wzc gene as previously described -arabinose (Sigma) at a final concentration of 1.0%. For the Δcps mutant, we deleted the chromosomal region from galF to wzi [Δ]. PCR fragments containing hns, mrkA and cps sequences flanking a kanamycin cassette were generated using gene-specific primer pairs  and are listed in Table g for 2 min and loaded into the LightCycler 480 instrument (Roche). Amplification was performed in triplicate wells for each sample analyzed. Control reactions with no template (water) and minus-reverse transcriptase (RNA) were run with all reactions. Real-time PCR analysis was performed using the following conditions: denaturation (95°C for 10 min); amplification and quantification repeated for 45 cycles ; melting curve ; and finally a cooling step at 40°C for 10 s. Melting curve analysis was performed after each run to confirm specificity of the primers. 16S rRNA was used as a reference gene for normalization and the relative gene expression was calculated using the 2−ΔCt method  were infected with the indicated strains of an LB broth overnight culture at a multiplicity of infection (MOI) of 100. Epithelial cells were grown in DMEM (Invitrogen) with 10% fetal bovine serum (FBS). After infection, eukaryotic cells were incubated in DMEM with no FBS for 2 h at 37°C under an atmosphere of 5% CO2. After the 2 h incubation period, cells were rinsed three times with PBS to remove unbound bacteria. For quantification of adherence, the cells were lysed with a solution of 0.1% Triton X-100. After homogenization, 10-fold serial dilutions were plated onto LB agar plates to determine total CFUs. The results shown are the mean of at least three experiments performed in triplicate on different days.Monolayers of HeLa (ATCC CCL-2) human cervix epithelial and A549 (ATCC CCL-185) human lung epithelial cell lines (7 × 105) were seeded into 24-well tissue culture plates. Bacteria were grown in 5 ml of LB broth to the exponential phase. Macrophages were infected with a MOI of 100 in a final volume of 1 ml RPMI 1640 tissue culture medium supplemented with 10% heat-inactivated FBS. To synchronize infection, plates were centrifuged at 200 g for 5 min. Plates were incubated at 37°C under an humidified 5% CO2 atmosphere. After 2 h, cells were rinsed three times with PBS and incubated for an additional 60 min with 1 ml of RPMI 1640 containing 10% FBS and gentamicin (100 μg/ml) to eliminate extracellular bacteria. Cells were then rinsed again three times with PBS and lysed with 0.1% Triton X-100. After homogenization, 10-fold serial dilutions were plated onto LB agar plates to determine total CFUs. Adherence of K. pneumoniae strains  to macrophages was performed as previously described  human monocytes (differentiated to macrophages with 200 nM of phorbol 12-myristate 13-acetate for 24 h) and RAW264.7 (ATCC TIB-71) murine macrophages (6 × 10600 using an enzyme-linked immunosorbent assay (ELISA) Multiskan plate reader.Adhesion to abiotic surface (polystyrene) was analyzed using 96-well plates as described previously . K. pneumoniae strains were homologous to H-NS proteins of enteric bacteria such as Salmonella, Yersinia, Shigella, and E. coli . By using the λ-red homolog recombinase . The growth in LB broth of K. pneumoniae strains was followed over a period of 8 h at 37 and 25°C. Growth of the Δhns mutant was slightly attenuated at 37°C, mainly in the exponential phase but reaching the stationary phase like the wild-type strain  similar to the ΔmrkA mutant, while this phenotype was counteracted by complementing the Δhns mutant with the pT3-H-NS plasmid  in the wild-type strain  regardless of the excess production of CPS . To determine the levels of mucoviscosity, we measured the supernatant of suspensions of wild-type, Δhns mutant and complemented Δhns mutant bacteria centrifuged at low speed. Indeed, the absence of H-NS resulted in higher mucoviscosity levels compared to the wild-type strain (~3-fold), while complementation restored the wild-type phenotype , indicating that increasing of the capsule in the hns background is dependent on capsular genes. Genetically, capsule-generating genes of K. pneumoniae are encoded in the cps cluster, including three transcriptional units, being galF, wzi, and manC the first genes for each promoter  and capsule-generating  genes in the wild-type and Δhns mutant at 37 and 25°C. At 25°C, the repressor effect of H-NS was higher than at 37°C, specifically 3.33-, 8.72-, and 9.66-fold for mrkA, fimA, and ecpA, respectively , while the complemented Δhns mutant showed an adherence that was similar to that of the wild-type strain  but not comparable to the Δhns mutant, suggesting that the phenotype observed with the Δhns mutant cannot be explained solely by the decrease of MrkA expression  and Δhns mutant (~263-fold) . Bacteria deficient in both hns and cps genes adhered in higher numbers to epithelial cells compared to those deficient in hns alone (~15-fold). However, the levels of adherence of the Δhns Δcps double mutant did not reach the numbers of the wild-type strain, indicating that CPS is partially involved in the low adherence observed with the Δhns single mutant . A crucial event in the pathogenesis of K. pneumoniae is the evasion of macrophage phagocytosis. We used THP-1 differentiated macrophages and RAW264.7 cells to evaluate the effect of H-NS deletion on phagocytosis. A Δhns mutant was considerably less phagocytized by macrophages (~55-fold) and the complemented strain was recovered in numbers similar to the wild-type strain . In contrast, a Δhns Δcps double mutant was phagocytosed in similar bacterial numbers as the Δhns single mutant. To determine if the low level of phagocytosis showed by the Δhns Δcps double mutant was due to the initial stage of adherence, we performed adherence assays using both macrophages cell lines. The Δhns and the Δhns Δcps mutants presented the same low levels of adherence to macrophages, indicating that the evasion of phagocytosis is mainly due to impaired adherence  Figure , supportt Figure . To discn Figure . Bacterie Figure . These oK. pneumoniae virulence features. The K. pneumoniae H-NS protein is homologous to other H-NS-like proteins of different enteropathogens such as E. coli, Salmonella, Yersinia, and Shigella. In terms of bacterial fitness, the absence of H-NS resulted in a slight defect in K. pneumoniae growth in LB broth at 37°C, but more evident at low temperature such as 25°C. As previously reported in other hns mutants, this could be due to dysregulated expression of non-related genes  and a capsular regulator (rcsA) were derepressed in the absence of H-NS. Moreover, the K. pneumoniae Δhns mutant was phagocytized in lower numbers as the wild-type strain, likely related to its high mucoviscosity which confers resistance to phagocytic uptake by macrophages , Gram-positive Lactobacillus rhamnosus ScaCBA pili and Streptococcus pneumoniae RrgA pili have been described to be required for the phagocytic uptake by macrophages  (Maurelli and Sansonetti, K. pneumoniae were differentially repressed at 37 and 25°C, showing that H-NS-mediated repression is higher at the lower temperatures likely encountered outside of the host.Change of temperature is an environmental condition that affects the oligomerization state of H-NS and subsequently its DNA-binding properties, being crucial for control of transcriptional regulation (Ono et al., K. pneumoniae. Our data show that similar to other pathogens, K. pneumoniae H-NS is a master regulator assuring optimal expression of both T3P and CPS whose uncontrolled expression may severely impact bacterial fitness.In summary, we have described for the first time the important role of H-NS in Conceived and designed the experiments: MA, JF RR, MJ, MA, MD. Performed the experiments: MA, JF, RR, MJ MD. Analyzed the data: MA, JT, JG, MA, MD. Wrote the paper: MA, KV, MA, MD.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A tumor model that Epstein-Barr virus (EBV) latent infection facilitated the tumorigenicity was previously established using the Maxi-EBV system. In the present approach, EBV-lost cell clones demonstrated significantly decreased tumorigenesis. On the other hand, the LMP1 gene in Maxi-EBV genome was replaced by that of nasopharyngeal carcinoma origin. The resultant cell line, 293–1/NL showed much lower malignancy than the original 293-EBV. The result was opposite to our expectation. The change of 293 sublineage cells for EBV harboring also got similar result. To seek the underlying reason, the copy number of EBV genome in all the cell lines was detected. The result indicated that 293-EBV contained about 4.5-fold higher EBV copies than 293–1/NL did. Parallel EBV genomes led to relatively stable copies in different 293 sublineages, suggesting the viral genome structure is a factor for the sustainability of EBV's copy number. Moreover, the LMP1 transcription in high copy-containing cells showed abnormally high level. Furthermore, the main LMP1-driven pathway, transcription factor NF-κB, was highly activated in high-copy cells. Here we first manifest by experimental model that the copy number of EBV latent genome correlates with the viral pathogenesis, which depends on the activation level of LMP1 and NF-κB. Overall, both the presence and amount of EBV genome are crucial for the viral oncogenicity. Epstein–Barr virus (EBV) is a ubiquitous human gammaherpesvirus that is the first confirmed oncogenic virus. It is highly associated with tumor development of both lymphoid and epithelial origins, including Burkitt's lymphomas and nasopharyngeal carcinoma (NPC) in which EBV genome can be found in almost all cells , 2. EBV EBV is a complex virus due to its cancer association, special life cycle and large genome with more than 170kb of double strand DNA . The etiEBV-encoded LMP1 is known as the viral oncoprotein. We ever analyzed the sequence from 21 strains of full-lenth LMP1 genes of NPC origin (N-LMP1) . Comparein vitro [NPC cell lines primarily containing EBV genome are commonly observed to undergo EBV-lost process during passages in vitro , 13. Thiin vitro . As demoin vitro . In thisin vitro, EBV genome was found to become lost from the cells gradually when without selection pressure. In order to know whether there would be changes in biological properties for the EBV-lost cells, clones of GFP-positive and GFP-negative were selected. They were identified by the detection of the gene transcription and protein expression of EBNA1. Here, the EBV genome-lost cell line, Lm, a mixture of 10 EBNA-negative clones showed a relative high ability of proliferation compared with the original 293–1 cells . In order to verify above result for N-LMP1 in genome analysis, these two EBV genomes containing B-LMP1 or N-LMP1 were introduced into 293–2 cells respectively. Two cell lines, C2089 and C22, were accordingly produced after hygromycin selection process  containing higher EBV copy number exhibited higher malignancy in nude mice. To see whether equal total EBV copies might have similar tumorigenicity, 4.5 × 10The expression of the oncoprotein LMP1 in the cells was detected Figure . The resLMP1 activates 3 main pathways, NF-κB, p38/MAPK and AP1 (c-Jun), and NF-κB is the most important –20. Rel in vitro for the lack of receptor CD21 on the cell membrane [To explore the causative role for EBV latent infection especially in epithelia cancers, cell and tumor models are important, but not satisfactory currently. The difficulty lies in several aspects of problem. One is that EBV is not able to infect epithelial cells directly membrane , 12. Themembrane , 9. It imembrane , it is din vitro [It is widely shared that EBV-positive epithelial cells lose EBV genome during passages in vitro , 12, 13.in vitro . This phin vitro . It has in vitro . As a faE.coli [As described previously , there iE.coli . Other'sE.coli . TherefoE.coli , 25. WheE.coli . In the 6 to 4 × 106 cells, and suitable range of observation time (e.g. 5 to 7 weeks) are included [Though the cell line 293 is not particularly relevant for EBV infection, we have shown that the 293-EBV tumor model in nude mice is useful for EBV oncogenicity analysis . NeverthLMP1 is an important functional protein in EBV oncogenicity , 27. In The C-terminal activating region (CTAR) domains of LMP1 activate three pathways, including the transcription factor NF-κB, p38/MAPK and c-Jun/AP-1 –20. We dIn this study, we present the first evidence by experimental model that the copy number of EBV is an important factor involved in the viral pathogenesis. We thus speculate that one cell with very low EBV copy number might not be transformed easily and develop a cancer. This may partly explain the fact that the ubiquitous virus is associated with only a minority of cancer. It has been noticed that EBV load in tumor tissues or blood is associated with the clinical progression and prognosis in both lymphoma and NPC –33. Our Taken together, the persistent latent infection of EBV genome in tumor cells is confirmed again an important factor for the tumorigenicity. Moreover, relative high copy number of EBV genome correlates with the development of cancer and should be considered into the cancer etiology. Although the precise role of EBV in the carcinogenic process remains to be fully understood, the oncoprotein LMP1 is a throughout crucial factor for the viral oncogenicity. The viral load and LMP1-driven NF-κB are important factors for the understanding of disease progression and EBV-targeted therapy.6 of cells [The plasmid p2089 (Maxi-EBV) which contains the complete EBV genome of B95–8 strain was kindly provided by Dr. W Hammerschmidt . The humof cells . Cell liof cells . The plaof cells . It coulOne full-length N-LMP1 gene  was chosen for this study. It had a homology of 88.8% compared with B-LMP1 . By homoThe EBV genome in 293-EBV could be lost during passages when cultured without selection pressure especially at low density [MTT assay was performed to measure the cell proliferation as described . BrieflyThis assay is a method for evaluating the ability of individual cell lines to grow in an anchorage-independent manner. Culture medium of 3ml containing 0.5% agar  and 10% fetal bovine serum was plated in six-well plates and allowed to solidify, creating a bottom layer. The cell suspension in 2 ml culture medium was combined with 0.3% agar, and plated onto the top of the bottom layer. The plates were examined twice per week till 30 days. The colonies were counted manually after 3 weeks. The experiment was repeated for three times.Cells of the amount as indicated were suspended in 200 ul growth medium and subcutaneously injected into the 4-week-old BALB/c nude mice . The mice were monitored and measured for the appearance and growth rate of tumors. Cell growth supernatant and cell lysates correspondingly were used for negative control. Preparing for histological examination and EBV detection, tumor tissues were dissected, fixed in 10% buffered formaldehyde, and embedded in paraffin.TM, Chicago, USA) was used as a loading control. The detection was performed on the ChemiDoc XRS+ Molecular Imager (Bio-Rad) using a Western Bright ECL kit . The Image J 2X software was used for the Gray-Scale quantification of the bands.Western Blotting was performed with a standard protocol. The general cell protein or nuclear proteins were extracted respectively. Proteins were separated on 10% SDS-polyacrylamide gels, electrophoretically transferred to polyvinylidene difluoride membranes , and detected using a mouse monoclonal antibody for anti-EBNA1  or anti-LMP1 . The β-actin antibody .Total RNA was isolated from the cells using TRIzol reagent. Protocol of a reverse transcription reaction was performed using the First Strand cDNA Synthesis Kit , and qPCR was performed using a Maxima SYBR Green qPCR Master Mix (2 ×) kit  according to the manufacturer's instructions. A CFX Multicolor Detection System (Bio-Rad) was employed for the detection. The primers in qPCR reactions for LMP1 were as: , and for β-actin were as: . The following program was performed using a two-step cycling protocol : an initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15s, 60°C for 60 s. Three parallel repeats were performed for each sample in each experiment, and the results were expressed as the mean of three independent experiments.General DNA was extracted from cells by using the General AllGen Kit  and quantified. Relative EBV copy number was detected according to the manufacturer's instructions of Epstein-Barr virus DNA Quantitative Fluorescence Diagnostic Kit . Specific PCR-fluorescence probe and a pair of specific primers for EBV conserved regions were provided in the kit. Real-time qPCR was performed. Two channels were detected. HEX channel was applied to detect the fluorescence of the internal standards to avoid false-negative results, requesting every sample to run an “s” curve which is also a basic request in real-time qPCR. The standard curve (R2 ≥ 0.98) was drawn according to the fluorescence signal changes (FAM channel) of quantitative reference substances (A/B/C/D), EBV-positive and EBV-negative controls. The EBV copy number concentration (copies/ml) of the samples was then calculated according to the standard curve. In the comparison of the relative copy number, the copy number of Raji which was standardized to 55 per cell, for Raji is accepted to contain 50–60 EBV latent genomes per cell . The expStatistical analysis was determined by one-way analysis of variance (ANOVA) test using the SPSS, version 11.0, program.